Question

1. You are studying Drosophila melanogaster and you hypothesize that a particular protein called “Eyeless” is essential for the eye to form.

1. You first decide to use the Gal4-UAS system to make sure that Eyeless is expressed in the eye. To do this you have found what you believe is the eye-specific enhancer for the Eyeless gene. You also decide to use GFP as a readout for protein production induced by the Eyeless enhancer. Describe how you will use the Gal4-UAS system and these components to determine if the Eyeless proteins might normally be expressed in the eye. Be sure to make it clear how the system works and how you have set it up. (12 points)

2. To determine if the Eyeless protein is required for the eye to form, you need to test what would happen if the fly develops without it! How could you use the Gal4-UAS system slightly differently to specifically inhibit the translation of the Eyeless mRNA in the eye to test this? (6 points)

Answer 1

Ans. -

The Gal4/UAS system is one of the most powerful tools for the study of cellular and developmental processes in Drosophila. Gal4 drivers can be used to induce targeted expression of dominant-negative and dominant-active proteins, histological markers, activity sensors, gene-specific dsRNAs, modulators of cell survival or proliferation, and other reagents. Demonstrate the induction of predicted eye phenotypes when expressing the dominant-negative EGF receptor, EGFRDN, or a dsRNA against Notch, NotchRNAi, in the developing eye disc.

The function of the eye specification factor Eyes absent or Eye in late retinal progenitors, shortly before they begin morphogenesis. Eye is still required in these late progenitors to promote eye formation, and show failure to induce the target gene atonal and consequent lack of neuron formation.

The GAL4 gene is placed under the control of a native gene promoter, or driver gene, while the UAS controls expression of a target gene. GAL4 is then only expressed in cells where the driver gene is usually active. In turn, GAL4 should only activate gene transcription where a UAS has been introduced.

GAL4 is a transcriptional activator that binds to UAS enhancer sequences found in DNA. It then recruits transcription machinery to the site to induce gene expression. Thus, genes and siRNA encoded downstream of the UAS sequence are only expressed where GAL4 is expressed.

The Drosophila Pax6 homolog Eyeless forms a complex, incompletely understood regulatory network with the Hedgehog, Decapentaplegic and Notch signaling pathways to control eye-specific gene expression. Here introduce the Drosophila melanogaster microRNA sponge (miR-SP) as a powerful transgenic technology to dissect the function of every microRNA with precise spatiotemporal resolution. miR-SPs can be used to characterize tissue-specific microRNA loss-of-function phenotypes, define the spatial regulation of their effectors and uncover interactions between microRNAs and other genes.